Recent developments in recombinant DNA procedures have created special problems in the purification of proteins from cell extracts and particularly in recovering cell proteins in useable forms. These recent developments in recombinant DNA procedures allow the synthesis of foreign proteins in host microorganisms, such as bacterial, yeast and animal cells. This is accomplished by transforming a host cell with a DNA sequence coding for the expression of a foreign protein. When the level of expression of the DNA sequence in a transformed host cell is high, a large amount of the foreign protein is produced within the host cell. Typically the cell sequesters most of these foreign proteins in inclusion bodies within the cytoplasm of the cell. The proteins sequestered in this manner are in the form of insoluble protein aggregates primarily composed of many monomers of the foreign protein bound together, typically through hydrophobic interactions.
The formation of these inclusion bodies containing the foreign protein normally would be helpful for the purification of the protein, because the inclusion bodies consist primarily of the foreign protein aggregates and, being insoluble, are readily isolated from the cell. However, in this insoluble, aggregate form, the protein is typically not in its preferred biologically active conformation such that the protein is capable of effecting its intended in vivo physiological responses.
Thus, purification of the protein in a biologically active form from the insoluble aggregates requires a means of solubilizing the protein aggregates in such a way to preserve, or to enable ultimate recovery of, the protein in a biologically active conformation.
The initial step in recovering the inclusion body contained proteins from transformed cells generally involves breakage of the cells through a combination of enzyme treatment and mechanical disruption to release the inclusion bodies. Because the protein aggregates contained in the inclusion bodies are insoluble, centrifugation of the resulting cellular material produces a membrane pellet containing a significant amount of the foreign protein, still in the form of insoluble aggregates. The pellet also contains lipids, lipopolysaccharides and traces of nucleic acid.
The next step typically used to recover the protein is to "solubilize" the insoluble protein aggregates. This may be accomplished by treating the membrane pellet with a strong chaotrope, e.g., guanidine hydrochloride, to denature and dissolve the protein. See, e.g., U.S. Pat. Nos. 4,511,502, 4,512,922, 4,518,526 and 4,620,948. "Solubilization" may also be effected using less stringent chatropes, e.g., urea, that "solubilize" but do not completely denature the desired protein. See, e.g., U.S. Pat. No. 4,652,630. If the protein contains more than one cysteine, it will usually have disulfide linkages in its structure. For these proteins, treatment with a reductant such as, e.g., dithiothreitol (DTT) to cleave the disulfide bonds may also be used during "solubilization" of the protein from the inclusion body. These chaotropes and reductants either alone or in combination will be referred to herein as "solubilizing agents".
When a protein is produced in vivo, its amino acid chains are "folded" into thermodynamically preferred three dimensional structures. Every protein has a unique folded conformation which is the most thermodynamically stable. This conformation is the protein's "native" conformation and gives the protein its characteristic biological activity. Factors influencing the protein's conformation ("conformation factors"), include steric interactions, charge interactions, Van der Waal forces, hydrophobic interactions and disulfide bond linkages between cysteine groups, if those are present in the protein.
Solubilizing agents disrupt the protein's conformation factors and "unfold" the protein to a degree that depends on the strength of the solubilizing agent. The greater the extent of the unfolding, the less degree of biological activity the protein likely displays. The resulting solubilized protein solution obtained after one or more of the above-described "solubilizations", thus comprises the foreign protein in some stage of unfolding, depending on the particular solubilizing treatment employed. To obtain a biologically active conformation of the desired protein, it must thus be "refolded". The solubilized protein solution also contains other soluble or solubilized phospholipids, lipopolysaccharides, proteins, and nucleic acids from the inclusion body and insoluble cellular debris. These must, of course, be removed either before or after refolding of the foreign protein.
A typical refolding method used to refold solubilized proteins involves diluting out the solubilizing agent with a large volume of diluent, generally a buffer. When the concentration of solubilizing agent is reduced to a dilution level where the protein's conformation factors begin to reassert themselves, the protein hopefully spontaneously refolds into a soluble, biologically active conformation. Depending on the protein, once this "optimal dilution level" is reached, refolding begins to occur within seconds or may take several minutes or longer.
Typically, the dilution is carried out in one step by mixing the solubilized protein solution with a diluent in an amount necessary to reach the optimal level of dilution. This dilution method is known as a "batch" dilution, drawing its name from the procedure of adding the diluent in one operation to the solubilized protein solution. Utilization of this batch method for refolding a solubilized protein has several disadvantages which are magnified when the refolding is carried out with the large volumes used in commercial scale purification procedures.
Because a solubilized protein solution generally has to be diluted with many times its volume of diluent to achieve at least some degree of refolding, the total volumes being handled at once in commercial protein purification methods can be very large, e.g., 5000 liters. This necessitates the use of large mixing chambers and special holding tanks and associated support systems, along with large amounts of chemicals for preparing the buffer diluents. Additionally, refolding proteins in large volumes by batch dilution may cause some reaggregation of the proteins, probably because the solutions at least initially present in batch dilutions are not homogeneous. This may result in a lowered net yield of refolded protein.
The non-homogenous solutions in batch dilutions result from the difficulty in rapidly achieving "ideal" mixing conditions in large volume solutions, i.e., conditions resulting in a homogenous solution without concentration gradients in solution. Ideal mixing conditions are a function of a solution's "mixing time". Mixing time is the time needed for the molecules in a droplet added to a solution to be dispersed evenly throughout the solution. Variables affecting mixing time include the volume of solution being mixed, size and configuration of the mixing chamber, the characteristics of the mixing device, and the location in the mixing chamber where the solutions are added. The larger the volumes of solution and the larger the size of the reaction vessel, the longer the mixing time and thus the longer that the mixture, e.g., of solubilized protein solution and diluent will not be homogenous.
A non-homogenous solution will have concentration gradients. These concentration gradients produce concurrent ionic strength variations in solution, which interfere with the charge interactions determining the conformation of the protein. Proteins refold into their native conformation through the repulsion and attraction of charges on their amino acid side chains and through the formation of cysteine-cysteine disulfide bonds. Variations in pH and ionic strength in turn may vary the charges and the attractive and repulsive forces, causing the protein to refold incorrectly or interact improperly with other nearby molecules. This phenomenon may decrease the net yield of correctly refolded proteins obtainable from a given volume of solubilized protein solution. Additionally, the use of large volumes makes it difficult to precisely control pH and ionic strength, resulting in less repeatable, less uniform yields. These problems have seriously hampered efforts to produce pure proteins in quantity at acceptable cost.
Conventional dilution procedures try to decrease the effect of these problems by using buffers as diluents to enhance ionic stability, the theory being that because buffers maintain isotonic concentrations, they will stabilize solution ionic strength and thus small concentration gradients will have less of an effect on solution ionic strength.
Conventional dilution methods do not utilize water as a diluent because it was commonly thought that water has no buffering effect. It would thus cause ionic strength variations in solution until completely mixed, thus causing excessive reaggregation of the proteins, and substantially decreasing the obtainable yield of a biologically active, soluble refolded protein (e.g., see U.S. Pat. No. 4,620,948). We have unexpectedly found that deionized water can be used as a diluent by using small volume mixing to maximize "ideal" mixing conditions.